In 1995, Fleischman et al. (40) determined the nucleotide sequence of Haemophilus influenzae Rd without prior restriction site mapping and cosmid cloning. This was a landmark event, signaling that automated DNA sequencing and analysis have matured into robust, highly efficient technologies promising to revolutionize the biological sciences. An important breakthrough that will greatly facilitate closure and proof- reading of the sequence which was reported by Dr. Mike Hunkapiller from ABI and Dr. Roe at the recent September Hilton Head DNA Sequencing conference was the results of experiments in which we successfully obtained sequence data in excess of 450 bases directly from several bacterial genomes. The genomic sequence of several other procaryotes have or will be completed shortly, including our own genomic sequencing of the important human pathogens Neisseria gonorrhoeae and Streptococcus pyogenes. Collectively, these studies will considerably enhance our understanding of procaryotic molecular biological processes, and provide new avenues for gene discovery and comparative genetics. From a practical standpoint, procaryotic genome sequencing will enhance our ability to understand processes occurring during the pathogenesis of infectious disease. These studies should provide new approaches for drug discovery, a necessity of increasing importance as microbial antibiotic resistance threatens our ability to treat bacterial infections. In this application, we propose to determine the nucleotide sequence of the genome of Staphylococcus aureus. This organism is a potent pathogen widely found in the human and animal environment. It is capable of producing upwards of 34 different extracellular proteins most of which have been shown to play a role in the pathogenicity of the organism or to enhance virulence. Although the incidence of infection in the general population is not well documented, it is generally accepted that S. aureus accounts for up to one-third of all nosocomial bacteremia. Other than the details of abscess formation, very little is actually of the pathogenesis of staphylococcal disease. Understanding the circuits (agr, sar and probably others) which control the expression of virulence related genes is paramount to understanding the genetic response of the organism to host generated signals. We propose that acquisition of the sequence of the genome of the Staphylococcus aureus strain 8325 will greatly facilitate understanding the mechanism of disease produced by this organism and closely related species. Therefore, as a single specific aim, we will sequence and annotate the 2.8 Mb genome of this important pathogen.